Synthetic oligonucleotides find numerous uses in molecular biology as probes for screening genomic and complementary DNA libraries, as primers for DNA synthesis, sequencing, and amplification, and in the study of DNA-protein interactions. In addition, oligonucleotide probes have proven useful for assaying in vitro gene expression using techniques of in situ hybridization.
Recent improvements in DNA sequencing methods, fluorescent labels, and detection systems have dramatically increased the use of fluorescently labeled oligonucleotides in all of these applications. Typically oligonucleotides are labeled with a fluorescent marker, either directly through a covalent linkage (e.g., a carbon linker), or indirectly whereby the oligonucleotide is bound to a molecule such as biotin or dioxigenin, which, is subsequently coupled to a fluorescently labeled binding moiety (e.g., streptavidin or a labeled monoclonal antibody).
These fluorescent labeling systems, however, suffer the disadvantage that the fluorescent complexes and their binding moieties are relatively large. The presence of large fluorescent labels and associated linkers may alter the mobility of the oligonucleotide, either through a gel as in sequencing, or through various compartments of a cell.
In addition, the presence of these markers alters the interaction of the oligonucleotide with other molecules either through chemical interactions or through steric hinderance. Thus the presence of these markers makes it difficult to study the interactions of DNA with other molecules such as proteins. The study of protein-DNA interactions is of profound interest as they involve some of the most fundamental mechanisms in biology. They include, for example, the progression of a DNA polymerase or reverse transcriptase along the length of the oligonucleotide, the activation of gene transcription as in the AP1 or steroid hormone pathway, or the insertion of viral DNA into the host genome as mediated by the HIV IN enzyme. For these reasons, it is desirable to obtain a fluorescent moiety analogous in structure to a pyrimidine or purine nucleotide and capable of being incorporated into an oligonucleotide. Such a moiety would preferably render the oligonucleotide molecule fluorescent without significantly altering the size or chemical properties of the oligonucleotide.
Numerous analogs of nucleotides are known. Among them are furanosyl pteridine derivatives. Methods of synthesizing these pteridine derivatives, which are structurally analogous to purine nucleotides, are well known. Indeed, a number of pteridine-derived analogs have been synthesized in the hope of discovering new biologically active compounds. Thus, Pfleiderer (U.S. Pat. No. 3,798,210 and U.S. Pat. No. 3,792,036) disclosed a number of pteridine-glycosides which possessed antibacterial and antiviral properties. Pfleiderer, however, did not investigate the fluorescent properties of these compounds.
Similarly, Schmidt et al., Chem. Ber. 106: 1952-1975 (1973) describe the ribosidation of a series of pteridine derivatives to produce structural analogs of the nucleoside guanosine, while Harris et al., Liebigs. Ann. Chem. 1457-1468 (1981), describe the synthesis of pteridine derivatives structurally analogous to adenosine. Again, neither reference describes measurements of the fluorescent properties of the nucleosides.
The synthesis of oligonucleotides incorporating lumazine derivatives has been described by Bannwarth et al., Helvetica Chimica Acta. 74: 1991-1999 (1991), Bannwarth et al., Helvetica Chimica Acta. 74: 2000-2007 (1991) and Bannwarth et al., (European Patent Application No. 0439036A2). Bannwarth et al. utilized the lumazine derivative in conjunction with a bathophenanthroline-ruthenium complex as an energy transfer system in which the lumazine derivative acted as an energy donor and the ruthenium complex acted as an energy receptor. Energy transfer occurred when the two compounds were brought into proximity resulting in fluorescence. The system provided a mechanism for studying the interaction of molecules bearing the two groups. The references, however, did not describe the use of a lumazine derivative alone in an oligonucleotide. In addition, Bannwarth recognized that a major disadvantage of the lumazine derivative was the " . . . relatively low extinction coefficient for the long wavelength absorption of the lumazine chromophore (.epsilon.=8900 m.sup.-1 cm.sup.-1 at 324 nm pH 6.9)." Bannwarth et al., Helv. Chim. Acta., 74: 1991-1999 (1991).
The present invention overcomes the limitations of these prior art compounds by providing a number of pteridine nucleotides which are analogous in structure to purine nucleotides, highly fluorescent under normal physiological conditions, and suitable for use in the chemical synthesis of oligonucleotides.